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ORIGINAL CONTRIBUTION
pH-responsive release of paclitaxel from
hydrazone-containingbiodegradable micelles
Peilan Qi1 & Yongqiang Bu1 & Jing Xu1 & Benkai Qin1
& Shujuan Luan1 & Shiyong Song1
Received: 8 August 2016 /Revised: 16 October 2016 /Accepted: 17
October 2016 /Published online: 5 November 2016# Springer-Verlag
Berlin Heidelberg 2016
Abstract Many tumor cells have acidic microenvironmentthat can
be exploited for the design of pH-responsive drugdelivery systems.
In this work, well-defined pH-sensitiveand biodegradable polymeric
micelles were prepared andevaluate as carrier of paclitaxel (PTX).
A diblock copolymerconstituting of a poly(ethylene glycol) (PEG)
and apolycaprolactone (PCL) segment linked by a
pH-sensitivehydrazone bond (Hyd), which was denoted as
mPEG-Hyd-PCL, was synthesized. The copolymer was assembled to
mi-celles with mean diameters about 100 nm. The mean diame-ters and
size distribution of the hydrazone-containing micellesincreased
obviously in mildly acidic environments while keptunchanged in the
neutral. No significant change in size wasfound on polymeric
micelles without hydrazone (mPEG-PCL). PTX was loaded into
micelles, and the anticancer drugreleased frommPEG-Hyd-PCL micelles
was promoted by theincreased acidity. In vitro cytotoxicity study
showed that thePTX-loaded mPEG-Hyd-PCLmicelles exhibited
significantlyenhanced cytotoxicity against HepG2 cells compared to
thenon-sensitive mPEG-PCL micelles. These results suggest
thathydrazone-containing copolymer micelles with pH sensitivityand
biodegradability show excellent potential as carriers ofanticancer
drugs.
Keywords Biodegradable copolymer . pH-sensitive .
Polymeric micelle . Controlled release
Introduction
Polymeric micelles have shown great potential in
hydrophobicanticancer drug delivery [1–3], while protecting them
duringcirculation, tomaximize the therapeutic efficacy
andminimizeside effects. Some micelle anticancer drug formulations,
e.g.,NK911®, SP1049C®, NK105®, and Genexol-PM®, haveadvanced to
clinical trials [4, 5]. Their nano-size andprolonged circulating
time facilitate the passive accumulationaround tumor tissues via
enhanced permeability and retention(EPR) effect. There are also
more and more functionalizedmicelles that were designed with active
targeting abilities tominimize systemic toxicity of therapeutic
agents [6]. It shouldbe noted that the effectiveness of a micelle
drug delivery sys-tem is determined by the control of not only
where the payloadshould be delivered but also when the payload
should be re-leased; e.g., drugs should be released quickly as soon
as thedrug delivery system arrives targeting sites. To this
end,stimulus-responsive micelle carriers have recently
attractedmuch attention because of their capability to release
incorpo-rated therapeutic agents in a responsive manner in
accordancewith the signals stemming from disease-associated
microen-vironment changes. Physical and chemical stimuli such as
pH[7–9], temperature [10], and reduction agents [11, 12]
wereexplored to trigger controlled drug release from the
micelles,determining when the drugs are to be released.
Among the many triggered release systems that are
beingevaluated, pH-sensitive drug delivery systems are
particularlyinteresting, because the acidity of many pathological
siteshave been well characterized. A pH gradient exists
betweennormal tissues and tumor which has acidic
microenvironment
Electronic supplementary material The online version of this
article(doi:10.1007/s00396-016-3968-6) contains supplementary
material,which is available to authorized users.
* Shiyong [email protected]
1 Present address: Institute of Pharmacy, Henan University,
NorthJinming Rd, Kaifeng 475004, China
Colloid Polym Sci (2017) 295:1–12DOI
10.1007/s00396-016-3968-6
http://dx.doi.org/10.1007/s00396-016-3968-6http://crossmark.crossref.org/dialog/?doi=10.1007/s00396-016-3968-6&domain=pdf
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[13]. The extracellular pH values in cancerous tissues are
low-er (5.7–7.0) than the normal blood pH of 7.4. pH gradients
canalso be found between the extracellular environment and
in-tracellular compartments such as endosomes and lysosomes(pH
4.5–6.5). As a result, the active anticancer drug could bereleased
effectively in mild acidic medium of the extracellularspace of the
tumor or in the acidic environment of endosomesor lysosomes
following cellular uptake.
Polymer-drug conjugate systems with acid-sensitive link-ages
between therapeutic molecule and macromolecules havebeen prepared
firstly. Ulbrich [7, 14] and Kataoka [15] syn-thesized block
copolymers conjugated with doxorubicin(DOX) via acid labile
hydrazone bond, which released conju-gated drug in an acidic
intracellular compartment upon cleav-age of hydrazone bonds. Jing
and his co-workers [16] havedeveloped micelles based hydrazone and
amide linkage forDOX and found higher pH sensitivity of
hydrazone-containing co-polymer. As acid labile bond, the
hydrazonebond formed between DOX and polymer hydrazines contain-ing
spacer has been most often studied. Other acid-sensitivebonds have
been seldom used. It is obvious that the conjuga-tion approach
requires appropriate bonding sites on both poly-mer and a drug
molecule to form acid labile bond, while otheranticancer drugs
(such as paclitaxel (PTX), camptothecin(CPT), and gemcitabine)
cannot form a hydrazone bond di-rectly as DOX.
An alternative way to form a pH-sensitive drug deliverysystem is
to physically entrap drugs into the hydrophobic coreof
pH-responsive polymeric micelles. The pH-sensitive bondis a part of
the copolymer, side chain or backbone. In this way,sufficient
structural change in the copolymer triggers boostdrug release upon
the cleavage of pH-sensitive bond in anacidic environment. Zhong
and his co-workers [17] synthe-s i z ed po ly ( e t hy l ene g lyco
l ) - b l ock -po ly (2 , 4 , 6
-trimethoxybenzylidene-pentaerythritolcarbonate) (PEG-PTMBPEC)
block copolymer and formulated pH-responsivebiodegradable micelles
and polymersomes. It was shown thatthe pH-dependent release of PTX
originated from the hydro-lysis of pendant acetal bonds on
hydrophobic PTMBPECblocks under endosomal pH condition. Yang and
his co-workers [18] constructed a pH-sensitive block
polymerPEG-b-C18. The hydrophilic block (PEG) and hydrophobicblock
(stearic acid) were connected by a Schiff base bond. Theacid labile
Schiff base bond cleaved under acidic condition,which resulted in
disassociation of micelle and consequentprompted drug release. It
is obvious that the pH-responsivedrug delivery system based on
cleave-disassociate-release(CDR) mechanism is applicably limited
not only to DOXbut also to PTX and CPT.
The profile of pH-triggered drug release is mainly deter-mined
by the sensitivity of acid labile bonds. The bondsshould hydrolyze
quickly at mild acidic environments andkeep stable at a pH of 7.4.
Acid labile linkages such as
hydrazone [7, 14–16], acetal [19, 20], orthoester [21,
22],citraconic amide [23], and Schiff base bonds [18] were
report-ed. They appeared in either side pendant chains or
backboneof the copolymers, acting either as drug-polymer linkages
ordiblock linkages. Among them, hydrazone bonds were mostlyexplored
for their easy synthesis, good stability, and moderatesensitivity.
Fu [24] was the first researcher who designeddiblock polymer with
hydrazone linkage. The copolymersshowed pH-dependent degradation
but not used as anticancerdrug carriers. The controllable
degradation and good compat-ibility of the copolymers were
approved. Georgiadou [25] andhis co-workers designed diblock
polymer with hydrazonelinkage. The copolymers showed pH-dependent
degradationbut not used as anticancer drug carriers.
PTX has demonstrated significant effect against a widerange of
tumors. However, some vital issues including poorwater solubility,
low bioavailability, and emergence of drugresistance largely
limited the application of PTX. The clinical-ly used PTX
formulation is a mixture of cremophor EL (alsocalled
polyoxyethylenated castor oil; cremorphor EL is a kindof non-ionic
surfactant, often used as a solubilizing agent ofinsoluble drug
use) and ethanol, which often leads to signifi-cant side effects
[26]. Due to the hydrophobic environment ofthe core of micelles,
water-insoluble drugs can easily be sol-ubilized and thus loaded
for delivery at the required targets[27]. So, PTX has been
formulated into nano-sized micellesand found enhanced antitumor
efficiency and minimized sideeffects. To realize temporally and
spatially controlled drugrelease, PTX has been seldom involved in a
pH-triggered drugdelivery system [28].
In this work, pH-sensitive copolymer was designed withhydrazone
bond as the acid labile linkage. Poly(ethylene gly-col) was used
for hydrophilic shell-forming block connectedwith hydrophobic
core-forming block, biodegradable poly(ε-caprolactone). The linkage
of the two blocks was hydrazonebond with adjacent conjugate group,
which was added to im-prove its chemical stability in air
atmosphere. Micelles basedon the copolymer were formed and PTX was
loaded. Perfectdrug delivery system with the characteristics of
intelligence,stealthiness, and biodegradability is expected, and
cleave-disassociate-release mechanism is hypothesized.
Experimental section
Materials
Methyl poly(ethylene glycol) (mPEG; Mn = 5000) was pur-chased
from Sigma-Aldrich and was dried for 24 h in a vac-uum oven at 50
°C before use. ε-Caprolactone (99 %,Aladdin) was dried with calcium
hydride by stirring for 24 hand distilled under reduced pressure
before use. Stannousoc tanoa te (Sn(Oct ) 2 ; 95 %, Sigma-Aldr ich)
, 4 -
2 Colloid Polym Sci (2017) 295:1–12
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carboxybenzaldehyde (98 %, Shanghai Darui),
N,N′-dicyclohexylcarbo-diimide (DCC; 98 % Shanghai Darui),
4-dimethylaminopyridine (DMAP; 98 % Shanghai Darui),methyl
4-hydroxybenzoate (98 %, Shanghai Darui), and hy-drazine hydrate
aqueous solution (80 %, Tianjin Kemiou)were used as received. All
organic solvents were analyticalreagents and used as received,
except that toluene was driedwith sodium method to get anhydrous
toluene.
Preparation of hydrazone-containing block
copolymermPEG-Hyd-PCL
Synthesis of aldehyde capped methyl poly(ethylene glycol)Methyl
poly(ethylene glycol) (mPEG-CHO) was synthesizedaccording to the
reported procedure with some modifications[18]. Briefly, mPEG (10
g) dissolved in dichloromethane(DCM) (150 mL) reacted with
4-carboxybenzaldehyde inthe presence of DCC and DMAP for 24 h at
room tempera-ture. Then, the solution was filtered and the filtrate
was con-centrated by rotary evaporation to remove DCM. The
rawproduct was dissolved in isopropanol and recrystallized atbelow
5 °C in a refrigerator. Resulted solid was washed withcold
isopropanol and ethyl ether subsequently. mPEG-CHOyellow powder was
obtained with a yield of 91.1 %, 1H NMR(400MHz, CDCl3), δ10.10
(Ar-CHO), 3.60 (–OCH2CH2O–),4.50 (–COOCCHO–), 3.80 (–COOCHC–), 3.37
(CH3O–),and 8.20 and 7.9 (aromatic protons).
Synthesis of 4-hydroxybenzoylhydrazine According to ref-erence
[29], methyl 4-hydroxybenzoate (9.2 g) and 17 mL ofhydrazine
hydrate (80 %) solution were mixed with smallamount of ethanol to
get a clear solution. The mixture wasrefluxed for 48 h and then
filtered. Washing it with ethanoltwo times yielded a white solid,
4-hydroxybenzoylhydrazine,which was dried in a vacuum oven for 24
h, 1H NMR(400 MHz, CDCl3), δ9.49 (Ar-OH), 7.60 and 6.70
(aromaticprotons), 9.90 (Ar-CONH–), and 4.36 (–N-NH2).
Synthesis of hydrazone-containing macroinitiator mPEG-Hyd-phenol
mPEG-CHO (1 g) reac ted wi th 4-hydroxybenzoichydrazine (0.15 g) in
DMF (10 mL) at60 °C for 24 h. mPEG-Hyd-phenol was isolated by
precipita-tion from cold ethyl ether and dried at 40 °C in vacuo,
1HNMR (400 MHz, CDCl3), δ8.40 (Ar-CH = N), 11.81 (Ar-CONH-N=),
10.16 (Ar-OH), and 3.60 (–OCH2CH2O–).
Synthesis of mPEG-Hyd-PCL block copolymer by ring-opening
polymerization Under a nitrogen atmosphere, thereis certain amount
of mPEG-Hyd-phenol and ε-caprolactone inthe presence of stannous
octanoate (100 μL) in anhydroustoluene. Polymers with different
molecular weights were syn-thesized by varying the ratio between
macroinitiator andmonomer. Typically, 0.304 g (0.057 mmol)
mPEG-Hyd-
phenol and 1.14 g (10.00 mmol) ε-caprolactone were placedin 20
mL anhydrous toluene. The mixture was stirred at100 °C in the
presence of stannous octanoate (100 μL) for24 h. Then, chloroform
was added to dissolve all the solid.The block copolymer was
isolated by precipitation from coldethyl ether and washed at least
five times with cold ethyl ether.It was dried at 40 °C in vacuo.
Block copolymers mPEG-Hyd-PCL with a target molecular weight of
25,000 were obtainedwith a yield of 59 %, 1H NMR (400 MHz, CDCl3),
δ3.60 (–OCH2CH2O–); δ8.40 (Ar-CH = N); 2.30, 1.60, 1.37, and
4.05(protons on poly(ε-caprolactone) part); and 6.91, 7.89, and8.09
(aromatic protons). Polymers of different molecularweights were
listed in Table 1.
Synthesis of mPEG-PCL block copolymer by ring-opening
polymerization mPEG-PCL copolymers withouthydrazone linkage were
also prepared as pH non-responsivecounterpart. The procedure was
the same as the mPEG-Hyd-PCL block copolymer except using mPEG as
initiator, 1HNMR (400 MHz, CDCl3), δ1.38, 1.64, 2.30, and 4.06
(pro-tons on poly(ε-caprolactone) part) and 3.64 (–OCH2CH2O–).
Characterization
Fourier transformed infrared spectroscopy (FTIR) wasperformed
using an AVATAR360 (Nicolet, USA) spec-trometer. 1H NMR spectra
were recorded on anAVANCE 400 spectrometer (Brucker, Switzerland)
oper-ating at 400 MHz using deuterated chloroform or deuter-ated
dimethyl sulfoxide as solvents. Chemical shift wascalibrated
against residual solvent signals. The molecularweight and
polydispersity of the copolymers were deter-mined by a Damn Eos
(Wyatt, USA) gel permeationchromatograph (GPC) instrument equipped
withPhenogel 10E6A column and a OPTILAB rEXrefractive-index
detector. Measurements were performedusing tetrahydrofuran (THF) as
the eluent at a flow rateof 1.0 mL/min at 30 °C and a series of
narrow polysty-rene standards for the calibration of the columns.
Thesize of the micelles was determined by dynamic lightscattering
(DLS) at 25 °C using a Zetasizer Nano-ZS90(Malvern Instruments, UK)
equipped with a 633-nm He–Ne laser. The micelle suspension was
filtered through a0.22-μm syringe filter before measurements. The
amountof PTX was determined by high-performance
liquidchromatography (HPLC) (Shimadzu LC-20AT, Japan)with UV
detection at 227 nm using a mixture of aceto-nitrile and water (v/v
= 55/45) as a mobile phase and aBDS HYPERSIL C18 (4.6 × 250 mm, 5
μm) column.Transmission electron microscopy (TEM) was
performedusing a JEM-100CX II TEM. The samples were preparedby
dropping 10 μL of micelle dispersion on the coppergrid and dried in
air.
Colloid Polym Sci (2017) 295:1–12 3
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Micelle formation and pH-triggered change of micelle size
Micelles were prepared using a simple solvent evapora-tion
method. mPEG-Hyd-PCL or mPEG-PCL (20 mg)was dissolved in acetone (1
mL). Under stirring, thesolution was added into 20 mL pure water by
dropwise.The resulting suspension was stirred at room tempera-ture
for 24 h. The resulting micelles were filteredthrough 0.22-μm
syringe filter, and the size was deter-mined by DLS. Three 10-mL
aliquots were taken fromfreshly prepared micelle dispersions, and
the pH of mi-celle dispersions was adjusted to pH 5.0 and pH
4.0using acetate buffer or maintained at pH 7.4 using phos-phate
buffer. The sizes were measured on DLS after 24-h incubation at 37
°C with shaking.
Critical micelle concentration measurement
Critical micelle concentration (CMC) was determinedusing pyrene
as a fluorescence probe. Ten vessels wereadded 0.5 mL stock
solution of pyrene in acetone(6.0 × 10−6 mol/L), respectively. They
were left in thedark and dried in air. Then, 5-mL micelle
dispersionwith different polymer concentration was added into
each vessel. The concentration varied from 1.0 × 10−10
to 0.1 mg/mL, and the pyrene concentration was fixedat 0.55 μM.
Fluorescence spectra were recorded usingan F-7000 (Hitachi, Japan)
fluorescence spectrometerand an excitation wavelength of 335 nm.
Fluorescenceemissions at 375 and 386 nm were monitored. TheCMC was
estimated at the cross-point when extrapolat-ing the intensity
ratio I375/I386 at low- and high-concentration regions.
Encapsulation and release of PTX
PTX-loaded micelles were prepared by a solvent
evaporationmethod. Typically, copolymers (20 mg) and PTX (2 mg)
weredissolved in 1 mL acetone. Under magnetic stirring, the
solu-tion was added by dropwise into 40 mL pure water at
roomtemperature. PTX-loaded micelles were formed after evapo-ration
of acetone. The dispersion was filtered through0.22-μm syringe
filter to remove undissolved PTX, thensealed, and stored in
refrigerator. To determine drug-loadingcontent (DLC), 1 mL of
PTX-loaded micelle dispersion wasfreeze-dried, the residue was
dissolved in acetonitrile, and theamount of PTX was determined by
HPLC. DLC was deter-mined according to the following formula:
DLC wt%ð Þ ¼ weight of loaded drug=total weight of loaded drug
and polymerð Þ � 100%
The in vitro release of PTX from micelles was investigatedat 37
°C under three conditions. Three aliquots (2 mL) ofPTX-loaded
micelle suspension were put into three dialysisbag with a molecular
weight cutoff (MWCO) of 8000–14,000,respectively. The bags were
sealed and immersed in the fol-lowing three 20-mL different
buffers: acetate buffer (0.01 M,pH 4), acetate buffer (0.01 M, pH
5), and PBS (0.01 M, pH7.4). There were 0.5 wt% Tween 80 in all the
buffers. Atdesired time intervals, 2 mL of the release medium was
takenout and replenished with an equal volume of fresh medium.
The concentration of PTXwas determined byHPLCmeasure-ments.
Cumulative release was calculated according to fol-lowing
formula:
Er ¼Ve
Xn−1
1
Ci þ V0Cnmdrug
In this equation, Er is the cumulative release of PTX (%),Veis
the volume to be taken every time (mL), V0 is the volume of
Table 1 Synthesis of mPEG-Hyd-PCL Polymer (Mi/Mm)
a Yields (%) Mnb (kg/mol) Mnc (kg/mol) PDId CMCe (mg/L)
mPEG-Hyd-PCL25K 1:175 59 28.2 15.9 1.840 0.603
mPEG-Hyd-PCL35K 1:260 67 36.2 19.3 1.791 0.288
mPEG-Hyd-PCL45K 1:350 68 45.2 24.6 1.683 0.191
a Feed ratios in mole between initiator and monomerb Calculated
from 1H NMR spectrac GPC resultsd PDI polydispersity indexe
Critical micelle concentration determined using pyrene as a
fluorescent probe
4 Colloid Polym Sci (2017) 295:1–12
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medium (mL), Ci is the concentration when certain volume isto be
taken (μg/mL), mdrug is the total mass of PTX containedin the
release system (μg), and n is the sampling time.
In vitro toxicity evaluation
The cytotoxicity of the empty micelle, PTX-loaded mi-celles, and
free PTX were evaluated by MTT assay usingHepG2 and HeLa cells.
Cells were seeded onto a 96-wellplate at a density of 5 × 103 cells
per well in 100 μL of1640 medium containing 10 % FBS or Dulbecco’s
mod-ified Eagle medium (DMEM) containing 10 % FBS andincubated for
24 h (37 °C, 5 % CO2). The medium in
each well was then replaced by 100 μL of new 1640medium or DMEM
(containing 10 % FBS) containingvarious concentrations of micelles,
PTX-loaded micelles,or free PTX. The tests were conducted in
replicates offour for each concentration. Each sample was
performedin quintuplicate and the samples were calculated withcells
for 24 and 48 h, and the viability of cells wasmeasured using the
methylthiazoletetrazolium method.In brief, 100 μL
methylthiazoletetrazolium solutions(0.5 mg/mL in culture medium)
were added to each well.The cells were incubated for 4 h, and then,
100 μL ofDMSO was added to dissolve the resulting purple crys-tals.
The formazan crystals were dissolved in dimethyl
H3CO
O
H113
CHO
+
DCC/DMAP
H3CO
O
113
O
CHO
CH2CH2
OCH3
HO
O
NHNH2
HO
O
HO
O
+
H3CO
O
113
O
H
C NNH
O
OH
H3CO
O
113
O
H
C NNH
O
O
O
H
O
n
N2H4.H
2O
O
O
DMF
Scheme 1 Synthesis pathway ofpH-sensitive mPEG-Hyd-PCLblock
copolymer
Colloid Polym Sci (2017) 295:1–12 5
-
sulfoxide, and the absorbance correlatable with the num-ber of
viable cells was measured using a thermo micro-plate reader at 570
nm. Cells cultured in DMEM mediumor 1640 medium containing 10 % FBS
(without micelles)were used as controls. Cell viability (%) was
calculatedby the following equation [30]:
Cell viability %ð Þ ¼ Asample=Acontrol� �� 100
where Asample and Acontrol denote the absorbance of thesample
well and control well, respectively. Data are pre-sented as average
SD ± (n = 3).
Result and discussion
Synthesis of hydrazone-containing block copolymers
Placing acid labile hydrazone bond onto backbone but not
sidechain of a copolymer is the main point of the current work.The
hydrazone-containing diblock polymer was not preparedby direct
coupling of polymers with reacting end groups.Preparation was
started with hydrazone-containingmacroinitiator and followed by a
ring-opening polymeriza-tion. The synthesis of the diblock
copolymer is illustrated inScheme 1.
As shown in Scheme 1, mPEG-CHO was firstly formedby
esterification of mPEG with 4-carboxybenzaldehyde.
Then, 4-hydroxybenzoichydrazide was synthesized byhydrazinolysis
of methyl 4-hydroxybenzoate, which bearhydrazine group (–NH2HN2).
Hydrazone containingmacroinitiator was obtained by coupling
reaction betweenmPEG-CHO and 4 - hyd r oxyben zo i c hyd r a z i d
e .Subsequently, hydrazone containing the copolymermPEG-Hyd-PCL was
synthesized via ring-opening poly-merization with the initiator
which has a benzyl hydroxylgroup. The 1H NMR spectra of mPEG-CHO
(Fig. 1a) showsignal characteristic of δ10.10 (Ar-CHO), 3.60
(–OCH2CH2O–), 4.50 (–COOCCHO–), 3.80 (–COOCHC–),3.37 (CH3O–), and
8.20 and 7.9 (aromatic protons).Through the above analysis, we can
confirm the successof the synthesis of mPEG-CHO. 1H NMR showed
thatthe macroinitiator mPEG-Hyd-phenol was successfullysynthesized,
as revealed by presence of signal δ8.40attributable to hydrazone
protons (Ar-CH = N)(Fig. 1b). The 1H NMR spectra of
mPEG-Hyd-PCL(Fig. 1c) show clearly signal characteristic of
PEG(δ3.60) and poly(ε-caprolactone) (δ1.37, 1.60, 2.30,4.05). And
most importantly, characteristic signals forproton on hydrazone
bond (δ8.40) and aromatic protons(δ6.91, 7.89, 8.09) were found on
the spectra. mPEG-PCL which has no pH-sensitive linkage was
synthesizedas control.
The molecular weights of synthesized polymers aregiven in Table
1. mPEG-Hyd-PCL25K, mPEG-Hyd-PCL35K, and mPEG-Hyd-PCL45K were
designed basedon feed ratios (1:175, 1:126, and 1:350,
respectively)
Fig. 1 The 1H NMR spectra ofcopolymer mPEG-CHO
(a),mPEG-Hyd-phenol (b), andmPEG-Hyd-PCL (c)
6 Colloid Polym Sci (2017) 295:1–12
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between initiator and monomer, and the yields were 59,67, and 68
%, respectively. Their hydrophilic segmenthad the same molecular
weight (Mn = 5267 g/mol),while their hydrophobic block varied in
molecularweight. Their real molecular weights were calculatedbased
on 1H NMR. The integral ratio between reso-nances at δ4.05 (one of
methylene protons on PCL)and 3.60 (methoxy proton of PEG) was used
to calcu-
late. It was found that the values based on 1H NMRspectra were
almost consistent with the theoretical ones.Additionally, GPC
measurements revealed a unimodaldistribution with Mn of 15.9, 19.3,
and 24.6 kg/mol,respectively (polystyrene standards) and
polydispersityindices (PDIs) of 1.840, 1.791, and 1.683 (Fig. S1
inSupplementary Materials). So, well-defined mPEG-Hyd-PCL block
copolymer was successfully synthesized.
Formation and pH-triggered size change of micelles
The amphiphilic mPEG-Hyd-PCL polymers can be self-assembled into
micelles in aqueous solution by a solventevaporation method. The
particle size of the micelles wascharacterized by DLS. As shown in
Fig. 2a and Table 2,the micelles have diameters ranged from 105 to
121 nmand increase with their molecular weight. The CMCs ofthe
polymeric micelles were 6.03 × 10−4, 2.88 × 10−4, and1.91 × 10−4
mg/mL for mPEG-Hyd-PCL (25 K), mPEG-Hyd-PCL (35 k), and
mPEG-Hyd-PCLA (45 k), respec-tively, determined by fluorescence
measurements usingpyrene as a probe (Fig. S2 in Supplementary
Materials).The CMC values were calculated from the intensity
ratioof bands at 375 and 386 (I375/I386) (Fig. S3 inSupplementary
Materials). It was found that the CMC ofthe polymers decreased from
mPEG-Hyd-PCL25K tomPEG-Hyd-PCL45K, which originated from the
in-creased hydrophobic interaction of micelle core. The mi-celles
had a relatively low CMC, indicating the excellentstability to keep
their construct under the in vivo dilutedconditions, which will be
critical for the efficient deliveryof drugs to tumors.
The average particle size of the drug-loaded micelleswas smaller
than that of the blank micelles (Fig. 2b andTable 2). The
incorporation of PTX into the core ofmicelles may enhance the van
der Waals force of thehydrophobic segments of the micelles,
resulting in amore compact structure and a decrease of particle
size[31]. As is known to all, nanoparticles below 200 nmcan
accumulate in tumor tissue via the enhanced perme-ability and
retention (EPR) effect [32, 33]; therefore,mPEG-Hyd-PCL micelle
drug delivery system below100 nm in size would effectively reach
lesion sites
100010010
100010010
0
5
10
15
20
25
In
te
ns
ity
(%
)
Size (d.nm)
mPEG-Hyd-PCL25K
mPEG-Hyd-PCL35K
mPEG-Hyd-PCL45K
0
5
10
15
20
25
In
te
ns
ity
(%
)
Size (d.nm)
mPEG-Hyd-PCL25K-PTX
mPEG-Hyd-PCL35K-PTX
mPEG-Hyd-PCL45K-PTX
a
b
Fig. 2 The particle size of different molecular weight of
mPEG-Hyd-PCLblock copolymer micelles (a) and PTX-loaded polymeric
micelles (b)
Table 2 Average sizes of blankmicelles and drug-loaded micelles
Blank micelles Size (d nm) PDI PTX-loaded micelles Size (d nm)
PDI
mPEG-Hyd-PCL25K 105 0.077 mPEG-Hyd-PCL25K-PTX 86 0.072
mPEG-Hyd-PCL35K 110 0.099 mPEG-Hyd-PCL35K-PTX 96 0.018
mPEG-Hyd-PCL45K 121 0.104 mPEG-Hyd-PCL45K-PTX 102 0.088
Colloid Polym Sci (2017) 295:1–12 7
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and achieve the goal of pH-controlled drug delivery. Asshown in
Fig. 3, the morphology of PTX-loaded mPEG-Hyd-PCL35K polymer
micelles was observed by TEM.The drug-loaded micelles have a
spherical core-shellstructure, and the particle size of the
micelles is about100 nm, which is consistent with the result of
DLSmeasurement. And, the drug-loading content of mPEG-Hyd-PCL35K
was about 2.7 %.
The evolution of micelle sizes responsive to pH wasmonitored by
DLS measurements. The size changes ofmicelles with or without
hydrazone bonds in response todifferent pH were illustrated in Fig.
4. As shown inFig. 4a, the size distribution of
hydrazone-containingmPEG-Hyd-PCL35K micelles underwent obvious
changesin pH 4.0, pH 5.0 conditions, and the multiple
peaksappeared; there was obvious turbidity and sedimentationwhile
keeping stable in pH 7.4. The appearance ofmulti-scale
nanoparticles was resulted from the
0
20
40
60
80
100
Cu
mu
la
tiv
e re
le
as
e(%
)
Time/h
pH4.0
pH5.0
pH7.4
a
0 2 4 6 8 10 12 14 16 18 20 22 24
0 2 4 6 8 10 12 14 16 18 20 22 24
0
20
40
60
80
100
Cu
mu
la
tiv
e re
le
as
e(%
)
Time/h
pH4.0
pH5.0
pH7.4
b
Fig. 5 In vitro release of PTX from PTX-loaded mPEGHydPCL35K
(a)and mPEG-PCL35K (b)
0.1 1 10 100 1000 10000
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
In
ten
sity (%
)
Size(d.nm)
pH4.0
pH5.0
pH7.4
a
100010010
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
In
te
ns
ity
(%
)
Size(d.nm)
pH4.0
pH5.0
pH7.4
b
Fig. 4 Size change of micelles under different pH conditions
mPEG-Hyd-PCL35K (a) and mPEG-PCL35K (b)
Fig. 3 TEM images of PTX-loaded mPEG-Hyd-PCL35K
polymermicelles
8 Colloid Polym Sci (2017) 295:1–12
-
decomposition of the pH-sensitive micelles. The aggrega-tion of
PCL blocks which are insoluble in water makesthe larger particles
of 1000 nm above. In contrast, mPEG-PCL35K micelles without
hydrazone bonds kept un-changed under all pH conditions (Fig. 4b).
The pH-sensitive micelle drug delivery system will keep stableand
protect their payload from being cleared in bloodcirculation. When
they accumulate around acidic tumortissue via EPR effect or
internalized by tumor cell, boostrelease of drugs will occur to
enhance the therapeuticeffect.
pH-controlled release of PTX
The pH-trigged in vitro drug release behaviors were studied
atpHs 4.0, 5.0, and 7.4. As shown in Fig. 5, PTX released
fromPTX-loaded mPEG-Hyd-PCL35K micelles at physiological
pH was about ca. 40 % in 24 h. The release rate was
signifi-cantly accelerated at pH 5.0 and 4.0, with accumulated
releaseabove 80 % in 24 h, respectively. The system showed
higherdrug release rate at acidic environment than at
physiologicalmedium, that is because acid labile characteristic of
hydrazonebond endowed the polymeric mPEG-Hyd-PCL35K micelleswith
pH-controlled drug release profile. To compare the re-lease curves
(Fig. 5a, b), it can be found that PTX-loadedmPEG-PCL35K polymeric
micelles without hydrazone bond,at a pH of 4, 5, and 7.4, all
showed a consistent trend ofrelease, and under different pH
conditions, all the cumulativerelease was about 45 % in 24 h; there
was no pH-dependentrelease profile. Consequently, the molecular
structure of thepolymer-containing hydrazone bond can affect the
drugin vitro release behavior of micelles. As is well known,
theextracellular pH in tumors (pH 5.7–6.8) is lower than
thephysiological conditions (pH 7.4) [34]; therefore, the pH-
0
20
40
60
80
100
120
Cell viab
ility (%
)
mPEG-Hyd-PCL35K
mPEG-PCL35K
100502512.56.25
a
0
20
40
60
80
100
120
Cell viab
ility(%
)
mPEG-Hyd-PCL35K
mPEG-PCL35K
100502512.56.25
b
Micelle Concentration ( g/mL)
Micelle Concentration ( g/mL)
Fig. 6 The cytotoxicity of blank mPEG-PCL35K and mPEG-Hyd-PCL35K
micelles incubated with HeLa cells (a) and HepG2 cells (b)for 48
h
0
20
40
60
80
100
Free PTX
mPEG-HydPCL35K-PTX
mPEG-PCL35K-PTX
12060302015
0
20
40
60
80
100
Ce
ll v
ia
bility
(%
)C
ell v
ia
bility
(%
)
PTX Concentration ( g/L)
Free PTX
mPEG-Hyd-PCL35K-PTX
mPEG-PCL35K-PTX
15 20 30 60 120
PTX Concentration ( g/L)
a
b
Fig. 7 The cytotoxicity of PTX-loaded mPEG-Hyd-PCL35K,
PTX-loaded mPEG-PCL35K, and free PTX to HeLa cells at different
PTXconcentrations for 24 h (a) and 48 h (b)
Colloid Polym Sci (2017) 295:1–12 9
-
responsive release system might be applied in
anticancertherapies.
Cytotoxicity of the drug-loaded micelles
The toxicities of the blank micelles were tested in HeLa
andHepG2 cells using a MTT assay. The cell viabilities of HeLaand
HepG2 cells were above 90% for both blank mPEG-PCLand mPEG-Hyd-PCL
micelles, following 24- and 48-h incu-bation (Fig. 6),which means
that the blank micelles were re-markably non-toxic and
biocompatible up to a concentrationof 100 μg/mL.
To demonstrate the effect of hydrazone linkages
ofmPEG-Hyd-PCL35K on antitumor capability, in vitro cy-totoxicities
of free PTX, mPEG-PCL35K, and mPEG-Hyd-PCL35K micelles at a series
of equivalent concen-trations of PTX were evaluated against HeLa
and HepG2
cell lines. As shown in Figs. 7 and 8, all the formula-tions
showed a dose-dependent cell proliferation inhibi-tion behavior,
and prolonging incubation time from 24 to48 h led to more death of
tumor cells. Free PTX showedhigher in vitro toxicity to each
cancerous cell, comparedto the other two micelle formulations.
While, pH-sensi-tive, PTX-loaded mPEG-Hyd-PCL35K micelles weremore
toxic than PTX-loaded mPEG-PCL35K. Superiorcell-killing capability
of PTX-loaded mPEG-Hyd-PCL35K micelles may be due to the fact that
entry ofpH-sensitive micelles through endocytosis and drug re-lease
into the cytoplasm triggered by endosome pH arequick and efficient
processes [21].
Additionally, a significant difference in
proliferationinhibition between HeLa and HepG2 cells incubatedwith
the same formulation was found. The liver carci-noma cell line
HepG2 demonstrated less sensitivity topaclitaxel than the HeLa cell
line. The relative resis-tance to paclitaxel of the HepG2 cells
when comparedto the cancerous HeLa cells likely results from the
me-tabolism of PTX by liver cells to less toxic compounds[35,
36].
Conclusions
In summary, a new type of pH-sensitive biodegradablemPEG-Hyd-PCL
block copolymer was synthesized viar ing -open ing po lymer iza t i
on in i t i a t ed f rom ahydrazone-containing macroinitiator. The
resulted copol-ymer was composed of hydrophilic PEG with
fixedlength and hydrophobic PCL with different lengths,which could
be self-assembled into micelles withuniformed size and narrow size
distribution. The con-taining hydrazone provided the formed
micelles withpH-responsive properties of degradation into pieces
un-der acidic conditions. PTX was used as a model drugand was effec
t ive ly loaded into the micel les .Copolymers without hydrazone
bonds were also pre-pared as control. In vitro drug release results
showedthat PTX-loaded mPEG-Hyd-PCL presented a more rap-id and
complete drug release in the intracellular lyso-some environment
(pH 5.0). The results of in vitro cellassay revealed that the
micelles were non-toxic andgood biocompatibility. PTX-loaded
mPEG-Hyd-PCL mi-celles possessed higher antitumor activity to kill
theHeLa cells in comparison with PTX-loaded mPEG-PCL micelles.
Although further study in vivo is neces-sary to determine the
potential of the pH-sensitive mi-celles as drug delivery vehicle,
the initial results dem-onstrate they are promising for
drug-controlled releasein tumor therapy.
0
20
40
60
80
100
Ce
ll v
ia
bility
(%
)
PTX Concentration ( g/L)
Free PTX
mPEG-Hyd-PCL35K-PTX
mPEG-PCL35K-PTX
15 20 30 60 120
0
20
40
60
80
100
Ce
ll v
ia
bility
(%
)
Free PTX
mPEG-Hyd-PCL35K-PTX
mPEG-PCL35K-PTX
15 20 30 60 120
PTX Concentration ( g/L)
a
b
Fig. 8 The cytotoxicity of PTX-loaded mPEG-Hyd-PCL35K,
PTX-loaded mPEG-PCL35K, and free PTX to HepG2 cells at different
PTXconcentrations for 24 h (a) and 48 h (b)
10 Colloid Polym Sci (2017) 295:1–12
-
Acknowledgments This work was supported by the National
NaturalScience Foundation of China (NSFC 51375142), Research Fund
forExcellent Young College Teachers of Henan Province, and a key
projectfunded by the Education Department of Henan Province.
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict ofinterest.
References
1. Kataoka K, Harada A, Nagasaki Y (2001) Block copolymer
mi-celles for drug delivery: design, characterization and
biological sig-nificance. Adv Drug Deliv Rev 47:113–131
2. Sakai-Kato K, Nishiyama N, Kozaki M, Nakanishi T,Matsuda Y,
Hirano M, Hanada H, Hisada S, Onodera H,Harashima H, Matsumura Y,
Kataoka K, Goda Y, OkudaH, Kawanishi T (2015) General
considerations regardingthe in vitro and in vivo properties of
block copolymer mi-celle products and their evaluation. J Control
Release 210:76–83
3. Cho H, Lai TC, Tomoda K, Kwon GS (2015) Polymericmicel les
for mult i -drug del ivery in cancer. AAPSPharmSciTech 16:10–20
4. Cabral H, Kataoka K (2014) Progress of drug-loaded poly-meric
micelles into clinical studies. J Control Release 190:465–476
5. Eetezadi S, Ekdawi SN, Allen C (2015) The challenges
facingblock copolymer micelles for cancer therapy: in vivo barriers
andclinical translation. Adv Drug Deliv Rev 91:7–22
6. Makino J, Cabral H, Miura Y, Matsumoto Y, Wang M, Kinoh
H,Mochida Y, Nishiyama N, Kataoka K (2015) cRGD-installed
poly-meric micelles loading platinum anticancer drugs enable
coopera-tive treatment against lymph node metastasis. J Control
Release220:783–791
7. Hrubý M, Koňák Č, Ulbrich K (2005) Polymeric micellar
pH-sensitive drug delivery system for doxorubicin. J Control
Release103:137–148
8. Huang F, Cheng R, Meng F, Deng C, Zhong Z (2015)Micelles
based on acid degradable poly(acetal urethane):preparation,
pH-sensitivity, and triggered intracellular drugrelease.
Biomacromolecules 16:2228–2236
9. Ma Y, Fan X, Li L (2016) pH-sensitive polymeric micelles
formedby doxorubicin conjugated prodrugs for co-delivery of
doxorubicinand paclitaxel. Carbohydrate Polym 137:19–29
10. Akimoto J, NakayamaM, Okano T (2014)
Temperature-responsivepolymeric micelles for optimizing drug
targeting to solid tumors. JControl Release 193:2–8
11. Deng B, Ma P, Xie Y (2015) Reduction-sensitive
polymericnanocarriers in cancer therapy: a comprehensive
review.Nanoscale 7:12773–12795
12. Cheng R, Meng F, Deng C, Zhong Z (2015) Bioresponsive
poly-meric nanotherapeutics for targeted cancer chemotherapy.
NanoToday 10:656–670
13. Webb BA, Chimenti M, Jacobson MP, Barber DL
(2011)Dysregulated pH: a perfect storm for cancer progression. Nat
RevCancer 11:671–677
14. Ulbrich K, Šubr V (2010) Structural and chemical aspects
ofHPMA copolymers as drug carriers. Adv Drug Deliv Rev
62:150–166
15. Ba Y, Fukushima S, Harada A, Kataoka K (2003) Designof
environment-sensitive supramolecular assemblies for in-tracellular
drug delivery: polymeric micelles that are re-sponsive to
intracellular pH change. Angew Chem Int Ed42:4640–4643
16. Hu X, Liu S, Huang Y, Chen X, Jing X (2010) Biodegradable
blockcopolymer-doxorubicin conjugates via different linkages:
prepara-tion, characterization, and in vitro evaluation.
Biomacromolecules11:2094–2102
17. Chen W, Meng F, Cheng R, Zhong Z (2010)
pH-sensitivedegradable polymersomes for triggered release of
anticancerdrugs: a comparative study with micelles. J Control
Release142:40–46
18. Ding C, Gu J, Qu X, Yang Z (2009) Preparation of
multifunctionaldrug carrier for tumor-specific uptake and enhanced
intracellulardelivery through the conjugation of weak acid labile
linker.Bioconjug Chem 20:1163–1170
19. Gillies ER, Goodwin AP, Fréchet JMJ (2004) Acetals as
pH-sensitive linkages for drug delivery. Bioconjug Chem
15:1254–1263
20. Lu J, Li N, Xu Q, Ge J, Lu J, Xia X (2010) Acetals
moietycontained pH-sensitive amphiphilic copolymer self-assembly
usedfor drug carrier. Polymer 51:1709–1715
21. Tang R, Ji W, Panus D, Palumbo RN, Wang C (2011) Block
copol-ymer micelles with acid-labile ortho ester side-chains:
synthesis,characterization, and enhanced drug delivery to human
gliomacells. J Control Release 151:18–27
22. Cheng J, Ji R, Gao SJ, Du F, Li Z (2012) Facile synthesis of
acid-labile polymers with pendent ortho esters. Biomacromolecules
13:173–179
23. Cao J, Su T, Zhang L, Liu R, Wang G, He B, Gu Z
(2014)Polymeric micelles with citraconic amide as pH-sensitivebond
in backbone for anticancer drug delivery. Int J Pharm471:28–36
24. Zhou L, Yu L, Ding M, Li J, Tan H (2011) Synthesis
andcharacterization of pH-sensitive biodegradable polyurethanefor
potential drug delivery applications. Macromolecules44:857–864
25. Koutroumanis KP, Holdich RG, Georgiadou S (2013)
Synthesisand micellization of a pH-sensitive diblock copolymer for
drugdelivery. Int J Pharm 455:5–13
26. Xiong M, Tang L, Wang J (2011) Synthesis and properties
0fdiblock copolymers of poly(ethylene glycol) and
poly(2-methoxyethyl ethylene phosphate) for enhanced paclitaxel
solubil-ity. Acta Polym Sin 11:853–860
27. Ahmad Z, ShahA, SiddiqM, Kraatz HB (2014) Polymeric
micellesas drug delivery vehicles. RSC Adv 4:17028–17038
28. Xu Z, Zhu S, Wang M, Li Y, Shi P (2014) Delivery of
paclitaxelusing PEGylated graphene oxide as a nanocarrier. ACS Appl
MaterInterfaces 7:1355–1363
29. Zheng H, Hua D, Bai R (2007) Controlled/living free-radical
copo-lymerization of 4-(azidocarbonyl) phenyl methacrylate with
methylacrylate under 60Co γ-ray irradiation. Polymer Chemistry J
PolymSci 245:2609–2616
30. Ahmad Z, Tang Z, Shah A, Zhang D, Zhang Y (2014)
Cisplatinloaded methoxy poly (ethylene glycol)-block-poly
(L-glutamic ac-id-co-L-phenylalanine) nanoparticles against human
breast cancercell. Macromol Biosci 14:1337–1345
31. Hong BX, Ou JL, Li XF, Ye QJ, Feng CJ (2014)Preparation of
doxorubicin-loading sodium alginate nanopar-ticles and their in
vitro release. Central South Pharmacy 12:451–456
32. Maeda H, Matsumura Y (2011) EPR effect based drug design
andclinical outlook for enhanced cancer chemotherapy. Adv DrugDeliv
Rev 63:129–130
Colloid Polym Sci (2017) 295:1–12 11
-
33. Baish JW, Stylianopoulos T, Lanning RM, KamounWS, FukumuraD,
Munn LL, Jain RK (2011) Scaling rules for diffusive drug de-livery
in tumor and normal tissues. Proc Natl Acad Sci U S A
108:1799–1803
34. Engin K, Leeper DB, Cater JR, Thistlethwaite AJ, Tupchong
L,McFarlane JD (1995) Extracellular ph distribution in human
tu-mors. Int J Hypertherm 11:211–216
35. McAuliffe G, Roberts L, Roberts S (2002) Paclitaxel
administrationand its effects on clinically relevant human cancer
and non cancercell lines. Biotechnol Lett 24:959–964
36. Gagandeep S, Novikoff PM, Ott M, Gupta S (1999)
Paclitaxelshows cytotoxic activity in human hepatocellular
carcinoma celllines. Cancer Lett 136:109–118
12 Colloid Polym Sci (2017) 295:1–12
-
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pH-responsive release of paclitaxel from hydrazone-containing
biodegradable micellesAbstractIntroductionExperimental
sectionMaterialsPreparation of hydrazone-containing block copolymer
mPEG-Hyd-PCLCharacterizationMicelle formation and pH-triggered
change of micelle sizeCritical micelle concentration
measurementEncapsulation and release of PTXInvitro toxicity
evaluation
Result and discussionSynthesis of hydrazone-containing block
copolymersFormation and pH-triggered size change of
micellespH-controlled release of PTXCytotoxicity of the drug-loaded
micelles
ConclusionsReferences